Nearly 6.5 million people in the United States suffer from chronic wounds and annual treatment costs exceeds $25 billion (1). A wide variety of treatment options exist (2) but clinicians often try multiple treatments until find one that promotes further the healing process for a particular patient. The healing of wounds is conceptually thought to occur in distinct, overlapping stages—it begins with hemostasis, followed by a stage in which inflammation is prevalent, which in turn leads to a stage where formation of granulation tissue, cell proliferation, angiogenesis and re-epithelialization are dominant (3). Non-healing or slowly healing (chronic) wounds are typically characterized as being stuck in a persistent, inflammatory stage, unable to make a transition to the cell-proliferative stage. The evaluation of non-healing wounds is difficult, however, and is usually performed qualitatively based on gross visual examination (4, 5) that requires high skill and experience (6). It may also sometimes require biopsies at multiple locations of a wound followed by lengthy histological analyses (7, 8). A simple and fast method that characterizes healing progress in a wound has the potential to expedite clinical diagnosis, better inform treatment, ultimately reducing discomfort and promoting favorable wound healing outcomes for patients.
Several non-invasive, optical methods have shown promise for characterizing physical characteristics of wounds in vivo such as cutaneous blood flow and microcirculation (laser Doppler perfusion imaging), tissue structure (optical coherence tomography) and tissue temperature (thermal imaging) (9). However, biochemical characteristics of healing wounds can reliably be evaluated only by tissue biopsy followed by histology (10). In addition to being invasive, this method is tedious, laborious (11) and prone to subjective evaluation (12, 13). Raman spectroscopy holds the potential to simply and rapidly assess the biochemistry of a wound in situ and thus complement traditional histological approaches. Raman spectroscopy relies on the inelastic (Raman) scattering of photons incident on a material which absorb or release energy from vibrational modes of chemical bonds giving rise to frequency (i.e., energy) shifts in the photons (14). These ‘Raman’ shift frequencies are unique to individual chemical bonds and can help identify the chemical composition of a material. Various biological tissues have been analyzed by Raman spectroscopy and it has also been shown to be able to identify diseased tissue in ailments such as breast cancer, atherosclerosis and Alzheimer's disease (15). Past studies (16-18) have also measured Raman spectra of wounds but no study has demonstrated the use of the method to distinguish between different stages of wound healing in vivo. In particular, past analyses of the Raman spectra of wounds has been based on differences in individual Raman peaks or peak ratios that were assigned to specific proteins. This approach is of limited diagnostic utility with complex biological tissue because many different tissue components are made from common molecular structural units (amino-acids, sugars, fatty acids) and bonds.
Bacterial contamination and colonization occurs in almost all wounds and non-virulent strains of bacteria, in low numbers, have been shown to facilitate healing (19-20). However, underlying co-morbidities (diabetes, venous stasis, etc.), and host factors (poor blood perfusion, white blood cell dysfunction, hyperglycemia, etc.) can promote bacterial overgrowth (21) that delays healing. For example, infection by Staphylococcus aureus, Pseudomonas aeruginosa and β-hemolytic Streptococci has been implicated in delayed healing (21) via production of destructive enzymes and toxins (20). Elevated endotoxin production provokes increased production of pro-inflammatory cytokines such as IL-1 and tumor necrosis factor-α (22-23) which in turn increase matrix metalloproteases that degrade endogenous growth factors. Thus, the excessive inflammatory response provoked by bacteria is hypothesized to underlie formation of chronic wounds. In addition, the moist environment of a wound provides an ideal milieu for bacteria to form complex communities in a self-secreted extracellular polysaccharide matrix (EPS) known as a biofilm (20-21, 24-25). Over time, biofilms mature with complex microstructures such as water channels for transfer of nutrients, waste and intercellular signaling molecules (‘quorum sensing’ molecules) (26) creating protected microenvironments for microbial persistence.
Biofilm bacteria communicate via quorum-sensing and respond collectively to the environment, enhancing resistance to antimicrobial agents (including antibiotics, antiseptics) and host defense mechanisms (24). Their EPS layer provides a diffusion barrier to topical antibiotics and contains substances like alginate which scavenges free oxygen radicals, prevents phagocytosis, and binds cationic antibiotics such as aminoglycosides (27). Biofilm bacteria can also slow their metabolism to increase their survival rates (20). Furthermore, genotypic and phenotypic diversity within a biofilm makes it likely that some cells survive when challenged by external stresses (28). While characterizing biofilms in vitro is facile due to availability of various staining methods (29, 30), characterization in vivo is highly challenging because the complex background of host tissue makes visual assessment (by light or electron microscopy) difficult and because host tissues adsorb biofilm stains non-specifically. More elaborate methods (viz. in vitro culture of wound swabs (31, 32)) can be inaccurate and do not readily distinguish between biofilms versus planktonic microbes. Development of rapid and non-invasive methods for in situ identification of biofilms (e.g. using a Raman microspectroscopic approach) would allow clinicians to alter treatment strategies for biofilm eradication in real time, including physical debridement, antimicrobial wound dressings and use of various topical antimicrobials and antibiofilm agents (28). Furthermore, diagnosis of biofilm formation prior to maturation has the potential to substantially decrease the likelihood of dysregulation of the wound healing process and thus formation of chronic wounds.